Multi-specific binding conjugate, related pharmaceutical compositions and use

ABSTRACT

The present invention relates to a multi-specific binding conjugate, a related composition and use. The binding conjugate comprises binding moieties for two or more different receptors, co-receptors, antigens or cellular markers which moieties are coupled by a nanomaterial. The multi-specific binding conjugate can be used to modulate an immune response, treat or prevent a disease or condition (e.g., a cancer, autoimmune disease, pathogen infection or inflammatory disease).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/130,031 filedSep. 13, 2018, which is a continuation application of InternationalApplication No. PCT/CN2016/101984 filed Oct. 13, 2016, which claimspriority to International Application No. PCT/CN2016/080749 filed Apr.29, 2016. U.S. Ser. No. 16/130,031 is also a continuation-in-part ofU.S. Ser. No. 15/548,630 filed Aug. 3, 2017, now U.S. Pat. No.10,758,625, which is a 371 national phase of International ApplicationNo. PCT/CN2016/079307 filed Apr. 14, 2016 and claims priority to ChineseApplication No. 201510175130.3 filed Apr. 14, 2015. The contents of eachare all hereby incorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention directs to the field of medical immunology, andmore particularly to a multi-specific binding conjugate for inducing animmune response to a disease such as a cancer or infectious disease. Theinvention also directs to a composition, method and use related to theconjugate.

BACKGROUND

Some antibody conjugates, such as multi-specific antibodies (such asbispecific antibodies), can bind to two or more different antigens. Anumber of recombinant strategies have been developed to synthesize abispecific antibody, including a single-chain variable fragment(scFv)-derived form such as a diabody, a tandem diabody, BiTes (abispecific T cell adaptor) and DART (double affinity re-targeting) aswell as a form based on immunoglobulin G (IgG), such as Triomab, DVD-Ig(double variable domain antibody) and a two-in-one antibody. However, abispecific antibody may have poor pharmacokinetic and physicalproperties (such as immunogenicity) as well as difficulty inmanufacture. Therefore, improvement or alternative technique to suchprior art is required.

SUMMARY OF INVENTION

A multispecific binding conjugate is disclosed herein. The multispecificbinding conjugate described herein comprises binding moieties that bindto different receptors, co-receptors, antigens and/or cellular markerson different cells, and are coupled to a nanomaterial, such as ananoparticle.

The multispecific binding conjugate disclosed herein comprises at leastone first binding moiety and at least one second binding moiety coupledto a nanomaterial, such as a nanoparticle, wherein the first bindingmoiety binds to a receptor, co-receptor, antigen and/or cell marker onthe cytotoxic effector cell, and the second binding moiety binds to areceptor, co-receptor, antigen and/or cellular marker on the targetcell. The cytotoxic effector cell can be a cytotoxic cell. The cytotoxiccell can be a leukocyte. The leukocyte may be selected from the groupconsisting of a macrophage, neutrophil, eosinophil, NK cell, B cell, andT cell. The leukocyte can be a NK cell. The leukocyte may be a NK-like Tcell, i.e. NKT cell. The leukocyte may be a NK cell and T cell.

The target cell may be a cell to be cleared, and may be a diseased cell.For example, the target cell can be a cancer cell or a tumor cell. Thetarget cell can be a leukemia cell. The target cell can be a lymphocyte.The target cell can be a metastatic cell. The target cell can begenetically modified. The target cell can contain gene mutation. Thegene mutation can include oncogene mutation. The gene mutation can bemutation of a tumor-suppressing gene. The gene mutation can be mutationof the proto-oncogene. The target cell can be an inflammatory cell. Thetarget cell can be an infected cell. The target cell can be a pathogeniccell.

The multispecific binding conjugate is preferably bispecific, whereinone binding site is directed to a receptor, co-receptor, antigen and/orcellular marker on a leukocyte, and the second binding site binds to areceptor, co-receptor, antigen and/or cellular marker on a target cell,which may be a cancer cell or tumor cell or pathogen (i.e.,microorganism). The multispecific binding conjugate may also betrispecific, wherein one binding site (the first binding site) isdirected to a receptor, co-receptor, antigen and/or cellular marker on aleukocyte, and the second binding site binds to a receptor, co-receptor,antigen and/or cellular marker on a target cell, which may be a cancercell or tumor cell or pathogen (i.e., microorganism).

Exemplary receptor, co-receptor, antigen and/or cell marker on a T cellis selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD8,CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 and CD90. Exemplaryreceptor, co-receptor, antigen and/or cell marker expressed on a NK cellis selected from the group consisting of CD2, CD8, CD11b, CD16, CD38,CD56, CD57, ADAM17, KIR, KAR, KLR and CD137. Exemplary receptor,co-receptor, antigen and/or cell marker on a monocyte is selected fromthe group consisting of CD74, HLA-DR alpha chain, CD14, CD16, CD64 andCD89. Exemplary receptor, co-receptor, antigen and/or cell marker on aneutrophil is selected from the group consisting of CEACAM6, CEACAM8,CD16b, CD32a, CD89, CD177, CD11a, CD11b, and SLC44A2. The receptor,co-receptor, antigen and/or cell marker on a leukocyte may also be acheckpoint antigen, which may be selected from the group consisting ofLSECtin, CTLA4, PD1, PD-L1, LAG3, B7-H3, B7-H4, KIR and TIM3.Preferably, the receptor, co-receptor, antigen and/or cellular marker ona T cell is CD3, CD4 or CD8, or the receptor, co-receptor, antigenand/or cellular marker on a NK cell is CD16 or CD56.

The second binding moiety can bind to a receptor, co-receptor, antigenand/or cellular marker on a diseased cell or pathogen. In certainembodiments, the second binding moiety of the conjugate can target areceptor, co-receptor, antigen, and/or cellular marker on any type oftumor and any type of tumor cell. Exemplary type of cancer that can betargeted includes acute lymphoblastic leukemia, acute myelogenousleukemia, biliary cancer, breast cancer, cervical cancer, chroniclymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer,endometrial cancer, esophageal cancer, gastric cancer, head and neckcancer, Hodgkin's lymphoma, lung cancer, bone marrow thyroid cancer,non-Hodgkin's lymphoma, multiple myeloma, kidney cancer, ovarian cancer,pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer,bladder cancer, neuroendocrine cancer, gastrointestinal pancreatictumor, exocrine pancreatic cancer and Ewing sarcoma. However, a skilledartisan in the art will recognize that a known tumor-associated antigenis actually useful for any type of cancer.

The receptor, co-receptor, antigen and/or cell marker on a tumor cell orcancer cell can be selected from the group consisting of: carbonicanhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3, A33antibody-specific antigen, AFP, ART-4, B7, Ba 733, BAGE, BrE3-antigen,CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4,CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46,CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a,CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154,CDCl27, CDK-4/m, CDKN2A, CXCR4, CXCR7, CXCL12, HIF-1alpha,colon-specific antigen-p (CSAp), CEA(CEACAM5), CEACAM6, c-met, DAM,EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folatereceptor, G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunit, HER2/neu, HMGB-1,hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ,IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growthfactor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT,macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90,pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostateacid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25,RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72,tenascin, TRAIL receptor, TNF-alpha, Tn antigen, Thomson-Friedrichantigen, tumor necrosis antigen, TROP-2, VEGFR, ED-B fibronectin, WT-1,17-1A-antigen, complement factor C3, C3a, C3b, C5a, C5, angiogenesismarker, bc1-2, bc1-6, Kras, cMET, oncogene product.

The receptor, co-receptor, antigen and/or cell marker on a tumor cell orcancer cell may also a cell surface protein selected from the groupconsisting of cholecystokinin B Receptor, gonadotropin releasing hormonereceptor, somatostatin receptor 2, avb3 integrin, gastrin releasingpeptide receptor, neurokinin 1 receptor, melanocortin 1 receptor,neurotensin receptor, neuropeptide Y receptor and C-type lectin-likemolecule 1.

The pathogen may be selected from the group consisting of HIV virus,Mycobacterium tuberculosis, Streptococcus agalactiae,meticillin-resistant Staphylococcus aureus, Legionella pneumophila,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans,Histoplasma capsulatum, Hemophilis influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, Rabies virus, Influenza virus, Cytomegalovirus, Type Iherpes simplex virus, type II herpes simplex virus, human serumparvo-like virus, respiratory syncytial virus, varicella-zoster virus,hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human Tcell leukemia virus, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocytechoriomeningitis virus, Wart virus, Blue tongue virus, Sendai virus, Catleukemia virus, Reovirus, Poliovirus, Simian virus 40, mouse mammarytumor virus, Dengue fever virus, rubella virus, West Nile virus,Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosomarangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosomabrucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis,Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinellaspiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taeniasaginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasmaarthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasmalaidlawii, M. salivarium and M. pneumoniae.

The first and/or second binding moiety may be selected from the groupconsisting of a small molecule, a cell targeting molecule, a ligand, aprotein, a peptide, a peptoid, a DNA aptamer, a peptide nucleic acid, avitamin, a substrate or a substrate analog, an antibody or a fragmentthereof. The binding moiety is preferably a ligand, a receptor, anaptamer, an antibody or a fragment thereof, more preferably an antibodyor a fragment thereof.

The antibody used may be a murine antibody, sheep antibody and so on,chimeric antibody, humanized and human antibody or a fragment thereof.The antibody may also be a heavy chain antibody (such as a camelidantibody) or a fragment thereof. The antibody may have various isotypes,preferably human IgG1, IgG2, IgG3 or IgG4, more preferably thosecomprising human IgG1 hinge and constant region sequences. The antibodyor fragment thereof may be a chimeric human-mouse, humanized (humanframework and murine hypervariable (CDR) region) or fully humanantibody, and variant forms thereof, such as semi-IgG4 antibody(referred to as “single antibody”), as described by van der NeutKolfschoten et al. (Science 2007; 317: 1554-1557). More preferably, theantibody or fragment thereof can be designed or selected to comprise aconstant region sequence belonging to a particular allotype, which canreduce immunogenicity when administered to a human subject. The antibodyor fragment thereof used may also be a multispecific antibody (e.g., abispecific antibody) or a fragment thereof.

The nanomaterial may be a pharmaceutically acceptable nanomaterial,preferably a biodegradable nanomaterial, such as a nanoparticle. Morepreferably, the nanomaterial is any one of or a mixture of at least twoof poly(lactic acid-co-glycolic acid), polylactic acid,polycaprolactone, polybutylene succinate, polyaniline, polycarbonate,poly(glycolide-co-lactide) or poly(glycolide-co-caprolactone), mostpreferably poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid(PLA) and/or polycaprolactone (PCL). The average particle size of thenanoparticle may be, for example, about 10-990 nm, for example, about900 nm, about 850 nm, about 800 nm, about 750 nm, about 700 nm, about650 nm, about 600 nm, about 550 nm, about 500 nm, about 450 nm, about400 nm, about 350 nm, about 300 nm, about 250 nm, about 200 nm, about150 nm, about 100 nm or less, such as about 90 nm, about 80 nm, about 70nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, orabout 10 nm. Preferably, the nanoparticle has an average particle sizein the range of 10 to 500 nm, more preferably in the range of 10 to 300nm.

The present disclosure also provides a pharmaceutical compositioncomprising the multispecific binding conjugate disclosed herein. Thepharmaceutical composition may also comprise a leukocyte, preferably aNK cell, that binds to the multispecific binding conjugate. Thepharmaceutical composition may also contain other therapeutic agent fortreating the disease.

The present disclosure also provides a method of treating a disease orcondition comprising administering the conjugate or pharmaceuticalcomposition to a subject in need thereof.

The present disclosure also provides the use of the conjugate andpharmaceutical composition in the manufacture of a medicament fortreating or preventing the disease or condition.

The present disclosure also relates to the conjugate and pharmaceuticalcomposition for use in treating or preventing the disease or condition.

In certain embodiments, the conjugate and pharmaceutical compositiondisclosed herein can be used to treat cancer, such as the cancerdisclosed herein. Exemplary type of cancer includes acute lymphoblasticleukemia, acute myelogenous leukemia, biliary cancer, breast cancer,cervical cancer, chronic lymphocytic leukemia, chronic myelogenousleukemia, colorectal cancer, endometrial cancer, esophageal cancer,gastric cancer, head and neck cancer, Hodgkin's lymphoma, lung cancer,bone marrow thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma,kidney cancer, ovarian cancer, pancreatic cancer, glioma, melanoma,liver cancer, prostate cancer, bladder cancer, neuroendocrine cancer,gastrointestinal pancreatic tumor, exocrine pancreatic cancer and Ewingsarcoma. In other embodiments, a subject infected with a pathogenicorganism such as a bacterium, virus, or fungus can be treated using theconjugate or pharmaceutical composition disclosed herein. Exemplaryfungus that can be treated includes Microsporum, Trichophyton,Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans,Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidisor Candida albicans. Exemplary virus includes human immunodeficiencyvirus (HIV), herpes virus, cytomegalovirus, rabies virus, influenzavirus, human papilloma virus, hepatitis B virus, hepatitis C virus,Sendai virus, Cat leukemia virus, Reovirus, poliovirus, human serumparvo-like virus, Simian virus 40, respiratory syncytial virus, mousemammary tumor virus, varicella-zoster virus, dengue virus, rubellavirus, measles virus, adenovirus, human T cell leukemia virus,Epstein-Barr virus, murine leukemia virus, mumps virus, vesicularstomatitis virus, Sindbis virus, lymphocyte choriomeningitis virus orBlue tongue virus. Exemplary bacterium includes Bacillus anthracis,Streptococcus agalactiae, Legionella pneumophila, Streptococcuspyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, Mycobacterium tuberculosis or Mycoplasma.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view showing the assembly of the bispecificantibody of Example 1.

FIG. 2 shows the efficacy of the bispecific binding conjugateanti-CD56-PLGA-anti-MUC1 on killing cancer cells.

FIG. 3 shows the efficacy of the bispecific binding conjugateanti-CD16-PLGA-anti-MUC1 on killing cancer cells.

FIG. 4 shows the efficacy of the bispecific binding conjugateanti-CD16-PLGA-anti-CD19 on killing cancer cells.

FIG. 5 shows the efficacy of the bispecific binding conjugateanti-CD56-PLGA-anti-CD19 on killing cancer cells.

FIG. 6 shows the efficacy of the bispecific binding conjugateanti-CD16-PLGA-anti-CD20 on killing cancer cells.

FIG. 7 shows the efficacy of the bispecific binding conjugateanti-CD56-PLGA-anti-CD20 on killing cancer cells.

FIG. 8 shows the ratio of NK (CD3-CD56+) cells and CD3+ T cells in theprepared effector cells.

FIG. 9 shows the ability of the bispecific or trispecific bindingconjugate to aid NK in killing cancer cells.

FIG. 10 shows the efficacy of the prepared bispecific or trispecificbinding conjugate on inhibiting tumors in vivo.

DESCRIPTION

Before the method and composition of the present invention aredescribed, it should be understood that the invention is not limited tothe specific method or composition described, and thus may, of course,vary. It should also be understood that the terms used herein are onlyillustrative, but not limiting. The description of the embodiments isprovided for guiding a skilled artisan in the art to make and use thepresent invention, and is not intended to limit the scope of theinvention, and also is not intended to indicate that the followingexperiments are all or only the experiments performed. Efforts have beenmade to ensure the accuracy of the number used (e.g., amount,temperature, etc.), but some experimental errors and deviations shouldbe considered.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a skilled artisan in theart. Although any method and material similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, some potential and preferred methods and materials are nowdescribed. All publications mentioned herein are hereby incorporated byreference to disclose and describe the methods and/or materials relatedto the cited publications. It will be understood that in the event of aconflict, the present disclosure will control.

As will be apparent to a skilled artisan in the art upon reading thisdisclosure, each individual embodiment described and illustrated hereinhas discrete components and features that can be readily separated fromor combined with the features in several other embodiments, withoutdeparting the scope or spirit of the invention. Any of the enumeratedmethods can be implemented in the order of the recited events or in anyother order that is logically possible.

Unless stated otherwise, the article “a” or “an” as used in thedescription and in the appended claims means “one or more”.

Where a range of values is provided, it should be understood that eachintermediate value (up to one tenth of the lower limit unit) between theupper and lower limits of the range is also specifically disclosed,unless the context clearly dictates otherwise. The various smallerranges between any such values or intermediate values within the rangeas well as other such values or intermediate values within the range areincluded in the invention. The upper and lower limits of these smallerranges may be independently included in or excluded from the range andare subject to any particular exclusion in the range, wherein either orboth or none of the upper and lower limits included in each range withinthe smaller range is also included in the present invention. Where thestated range includes one or both of the limits, the scope of theexclusion of either or both of such limits is also included in theinvention.

A “therapeutic agent” is an atom, molecule or compound that can be usedto treat a disease. An example of a therapeutic agent include anantibody, antibody fragment, peptide, drug, toxin, enzyme, nuclease,hormone, immunomodulator, antisense oligonucleotide, small interferingRNA (siRNA), chelating agent, boron compound, photosensitizer, dye andradioisotope.

As used herein, an “antibody” refers to a full-length (i.e., naturallyoccurring or formed by the process of rearrangement of a normalimmunoglobulin gene segment) immunoglobulin molecule (e.g., an IgGantibody) or an immunoactive (i.e., specific binding) part of suchimmunoglobulin molecule (e.g., an antibody fragment). An “antibody”includes a monoclonal antibody, a polyclonal antibody, a bispecificantibody, a multispecific antibody, a murine antibody, a heavy chainantibody (such as a camelid antibody), a chimeric antibody, a humanizedantibody and a human antibody, and the like. A “heavy chain antibody” isan antibody that contains only a heavy chain and does not contain alight chain, such as a camelid antibody, a cartilaginous fish (such as ashark) antibody, and the like.

An “antibody fragment” is a part of an intact antibody, such as F(ab′)2,F(ab)₂, Fab′, Fab, Fv, scFv, dAb, and the like. Regardless of thestructure, the antibody fragment binds to the same antigen as recognizedby the full length antibody. For example, an antibody fragment comprisesan isolated fragment consisting of variable regions, such as an “Fv”fragment consisting of a heavy or light chain variable region or arecombinant single-chain polypeptide molecule (“scFv protein”) in whichthe light chain and heavy chain variable regions are joined by a peptidelinker. A “single-chain antibody” is often abbreviated as “scFv” andconsists of a polypeptide chain comprising V_(H) and V_(L) domains thatinteract with each other to form an antigen-binding site. The V_(H) andV_(L) domains are typically joined by a peptide having from 1 to 25amino acid residues. An antibody fragment also includes a bifunctionalantibody, a trifunctional antibody, and a single-domain antibody (dAb).An “antibody fragment” also includes a fragment of the heavy chainantibody, such as a single-domain antibody (sdAb) or a fragmentcomprising a variable region.

A “multispecific antibody” is an antibody that can simultaneously bindat least two targets with different structures (e.g., two differentantigens, two different epitopes on the same antigen). A “bispecificantibody” is an antibody that can simultaneously bind to two targetswith different structures.

If the amount administered is physiologically significant, the conjugateor composition described herein is described to be administered in a“therapeutically effective amount”. It is physiologically significant ifthe presence of an agent produces a detectable change in the physiologyof the subject. In a particular embodiment, if the presence of anantibody formulation causes an anti-cancer response or ameliorates asign and symptom of an infectious disease state, it is physiologicallysignificant. A physiologically significant effect may also be to elicita humoral and/or cellular immune response in the subject, therebycausing growth inhibition or death of a target cell.

Multispecific (e.g., Bispecific) Binding Conjugate

A “multispecific binding conjugate” means that the conjugate cansimultaneously bind to at least two targets with different structures(e.g., two different antigens, two different epitopes on the sameantigen). A “bispecific binding conjugate” means that the conjugate cansimultaneously bind to two targets with different structures.

Various embodiments are directed to a multispecific binding conjugatecomprising a first binding moiety binding to a leukocyte and a secondbinding moiety binding to a receptor, co-receptor, antigen and/orcellular marker on a cell associated with a disease or a cell to becleared, which moieties are coupled to a nanomaterial (such as ananoparticle). The first binding moiety and/or the second binding moietymay have one or more binding specificities, for example, may be one ormore monoclonal antibodies or fragments thereof, or a multispecificbinding moiety, such as a bispecific antibody or fragment thereof.

Exemplary receptor, co-receptor, antigen and/or cell marker on a T cellincludes CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L,CD44, CD45, CD69 and CD90. For a NK cell, other exemplary receptor,co-receptor, antigen and/or cell marker can be selected from the groupconsisting of CD8, CD16, CD56, CD57, ADAM17 and CD137. For a monocyte,exemplary receptor, co-receptor, antigen and/or cell marker may beselected from the group consisting of CD74, HLA-DR alpha chain, CD14,CD16, CD64 and CD89; and for a neutrophil, exemplary receptor,co-receptor, antigen and/or cell marker includes CEACAM6, CEACAM8,CD16b, CD32a, CD89, CD177, CD11a, CD11b and SLC44A2. In a preferredembodiment, the first binding moiety for NK binds to CD16 or CD56. Asdiscussed below, many examples of the disease-associated antigen, suchas a tumor-associated antigen (TAA) or an antigen expressed by apathogen, are known. Preferably, the TAA is MUC1, CD19, CD20, CD33,CD38, EGFR and HER2.

The binding moiety useful in the claimed conjugate is preferably anantibody or a fragment thereof. Techniques for preparing a monoclonalantibody that are actually directed against any target antigen are wellknown in the art. Known and/or commercially available antibodies canalso be used. For example, an antibody that may be used is selected fromthe group consisting of hR1 (anti-IGF-1R), hPAM4 (anti-mucin), KC4(anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP),hLL1 (anti-CD74), hLL2 (anti-CD22), RFB4 (anti-CD22), hMu-9 (anti-CSAp),hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7(anti-TROP-2), hMN-3 (anti-CEACAM6), CC49 (anti-TAG-72), J591(anti-PSMA), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX),infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab(anti-TNF-α), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20),panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20),GA101 (anti-CD20), trastuzumab (anti-HER2/neu), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-α4integrin), BWA-3 (anti-histone H2A/H4), LG2-1 (anti-histone H3), MRA12(anti-histone H1), PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B),LG2-2 (anti-histone H2B), P4/D10 (anti-gp120) and omalizumab (anti-IgE).

Exemplary antibody for the treatment of, for example, cancer includes,but are not limited to, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22),veltuzumab (hA20, anti-CD20), rituximab (anti-CD20), obinutuzumab(GA101, anti-CD20), ranibizumab (anti-PD1), nivolumab (anti-PD1),MK-3475 (anti-PD1), AMP-224 (anti-PD1), pidilizumab (anti-PD1), MDX-1105(anti-PD-L1), MEDI4736 (anti-PD-L1), MPDL3280A (anti-PD-L1), BMS-936559(anti-PD-L1), Ipilimumab (anti-CTLA4), Trevizumab (anti-CTL4A), RS7(anti-epithelium glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4 orKC4 (both as anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA,also known as CD66e or CEACAM5)), MN-15 or MN-3 (anti-CEACAM6), Mu-9(anti-colon specific antigen-p), Immu 31 (anti-alphafetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g. CC49), Tn, J591 or HuJ591(anti-PSMA (prostate specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), PAM4 (akakrivuzumab, anti-mucin), BWA-3 (anti-histone H2A/H4), LG2-1(anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B),LG11-2 (anti-histone H2B), LG2-2 (anti-histone H2B) and trastuzumab(anti-ErbB2). These antibodies are known in the art.

In certain embodiments, the receptor, co-receptor, antigen and/orcellular marker on the target cell can be a cancer cell receptor or acancer associated antigen, particularly selected from the groupconsisting of B cell line antigens (CD19, CD20, CD21, CD22, CD23, etc.),VEGF, VEGFR, EGFR, carcinoembryonic antigen (CEA), placental growthfactor (PlGF), tenascin, HER-2/neu, EGP-1, EGP-2, CD25, CD30, CD33,CD38, CD40, CD45, CD52, CD74, CD80, CD138, NCA66, CEACAM1, CEACAM6(carcinoembryonic antigen-associated cell adhesion molecule 6), MUC1,MUC2, MUC3, MUC4, MUC16, IL-6, alpha-fetoprotein (AFP), A3, CA125,colon-specific antigen-p (CSAp), folate receptor, HLA-DR, humanchorionic gonadotropin (HCG), Ia, EL-2, insulin-like growth factor (IGF)and IGF receptor, KS-1, Le(y), MAGE, necrotic antigen, PAM-4, prostaticacid phosphatase (PAP), Prl, prostate specific antigen (PSA), prostatespecific membrane antigen (PSMA), S100, T101, TAC, TAG72, TRAIL receptorand carbonic anhydrase IX.

MUC1 antigen is highly abnormally expressed on the surface of the cellof many tumors (90% of solid cancers such as liver cancer, lung cancer,breast cancer, esophageal cancer, gastric cancer, colorectal cancer,cervical cancer, kidney cancer, bladder cancer, etc.), making it apotential target molecule for tumor-targeted therapy.

B cell expresses various cell surface molecules during theirdifferentiation and proliferation. Examples include CD10, CD19, CD20,CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a,CD79b, CD80, CD81, CD82, CD83, CD84, CD85 and CD86 leukocyte surfacemarkers. These markers are generally considered to be therapeutictargets for the treatment of B cell disorders or diseases such as B cellmalignancy, autoimmune disease, and transplant rejection. Antibodiesthat specifically bind to these markers have been developed, and somehave been identified as therapeutic agents for the treatment of thediseases and disorders.

For multiple myeloma therapy, suitable targeting antibodies have beendescribed for, e.g. CD38 and CD138 (Stevenson, Mol Med 2006; 12(11-12):345-346; Tassone et al, Blood 2004; 104(12): 3688-96), CD74 (Stein etal, supra), CS1 (Tai et al, Blood 2008; 112(4): 1329-37) and CD40 (Taiet al, 2005; Cancer Res. 65(13):5898-5906).

CD74 antigen is highly expressed in B cell lymphoma (including multiplemyeloma) and leukemia, certain T cell lymphoma, melanoma, colon cancer,lung and kidney cancer, glioblastoma and some other cancers (Ong et al,Immunology 98: 296-302 (1999)). A review of the use of CD74 antibody intreating cancer is provided in Stein et al, Clin Cancer Res. Sep. 15,2007; 13 (18 Pt 2): 5556s-5563s, which is incorporated herein byreference. Preferred indications of an anti-CD74 antibody include, butare not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma,lung cancer, renal cancer, colon cancer, glioblastoma multiforme,histiocytoma, myeloid leukemia and multiple myeloma.

In another preferred embodiment, an antibody against a pathogen can beused, and such an antibody is known. Commercially available antibodiesagainst a variety of human pathogens (e.g., KPL, Inc., Gaithersburg,Md.) can be utilized, including antibodies against Staphylococcus aureus(Cat. No. 011-90-05), Streptococcus agalactiae (Cat. No. 011-90-08),Streptococcus pyogenes (Cat. No. 01-90-07), Helicobacter pylori (Cat.No. 01-93-94), Borrelia burgdorferi (Cat. No. 05-97-91), Escherichiacoli (Cat. No. 01-95-91; 01-95-96), Legionella (Cat. No. 01-90-03),Listeria (Cat. No. 01-90-90), Vibrio cholerae (Cat. No. 01-90-50),Shigella (Cat. No. 16-90-01) and Campylobacter (Cat. No. 01-92-93).

In a preferred embodiment, the pathogen is selected from the groupconsisting of HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, meticillin-resistant Staphylococcus aureus, Legionellapneumophila, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Rabies virus, Influenza virus,Cytomegalovirus, Type I herpes simplex virus, type II herpes simplexvirus, human serum parvo-like virus, respiratory syncytial virus,varicella-zoster virus, hepatitis B virus, hepatitis C virus, measlesvirus, adenovirus, human T cell leukemia virus, Epstein-Barr virus,murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbisvirus, lymphocyte choriomeningitis virus, Wart virus, Blue tongue virus,Sendai virus, Cat leukemia virus, Reovirus, Poliovirus, Simian virus 40,mouse mammary tumor virus, Dengue fever virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, as disclosed inU.S. Pat. No. 6,440,416. The examples of this patent are incorporatedherein by reference in their entirety.

Other antibodies that may be used include antibodies against infectiousdisease factors such as bacteria, viruses, mycoplasmas or otherpathogens. Many antibodies against such infectious agents are known inthe art, and any such known antibodies can be used in the claimedmethods and compositions. For example, the following antibodies can beused: an antibody against the gp120 glycoprotein antigen of humanimmunodeficiency virus I (HIV-1); antibody against malaria parasites,such as monoclonal antibody against sporozoite (circumsporozoiteantigen), antibody against schistosomulum surface antigen (Simpson etal, Parasitology, 83: 163-177, 1981; Smith et al, Parasitology, 84:83-91, 1982; Gryzch et al, J. Immunol., 129: 2739-2743, 1982; Zodda etal, J. Immunol. 129: 2326-2328, 1982; Dissous et al, J. Immunol., 129:2232-2234, 1982); antifungal antibody, such as anti-Sclerotiniascleroterum antibody (U.S. Pat. No. 7,910,702), anti-Glucose caprylicacid xylose mannan antibody (Zhong and Priofski, 1998, Clin Diag LabImmunol 5: 58-64); anti-candida antibody (Matthews and Burnie, 2001,Curr Opin Investig Drugs 2: 472-76) and anti-sphingoglycolipid antibody(Toledo et al, 2010, BMC Microbiol 10:47).

Suitable antibodies have been developed for most of the microorganisms(bacteria, viruses, protists, fungi, other parasites) that causeinfection in most people, and many have previously been used for invitro diagnostic purposes. These antibodies, as well as modifiedantibodies which can be produced by conventional methods, are suitablefor use in the present invention.

The nanomaterial for coupling the first binding moiety (e.g., anantibody or a fragment thereof) and the second binding moiety (e.g., anantibody or a fragment thereof) may be a pharmaceutically acceptablenanomaterial, preferably a biodegradable nanomaterial, more preferablyany one of or a mixture of at least two of poly(lactic acid-co-glycolicacid), polylactic acid, polycaprolactone, polybutylene succinate,polyaniline, polycarbonate, poly(glycolide-co-lactide) orpoly(glycolide-co-caprolactone), most preferably poly(lacticacid-co-glycolic acid) (PLGA), polylactic acid (PLA) and/orpolycaprolactone (PCL). These nanomaterials and methods for preparingthem are known in the art, for example, the nanomaterials can beprepared using the methods described below.

Preparation of the Conjugate of the Invention

The method for preparing the multispecific binding conjugate of thepresent invention is described below by taking a bispecific bindingconjugate comprising two antibodies coupled to a nanoparticle as anexample. The preparation method comprises the following steps:

(1) preparing, collecting and activating the nanomaterial;

(2) connecting the nanomaterial obtained in step (1) with a mixture ofthe first antibody moiety and the second antibody moiety.

In the step (1), the preparation of the nanomaterial comprises:completely dissolving the nanomaterial with a solvent, stirring, addingwater to form a uniform emulsion. The agitation may be carried out at arotation speed of 500 to 20,000 rpm/min, for example, the rotation speedmay be 500 rpm/min, 700 rpm/min, 800 rpm/min, 1000 rpm/min, 1100rpm/min, 1200 rpm/min, 1300 rpm/min, 1400 rpm/min, 1480 rpm/min, 1500rpm/min, 2000 rpm/min, 2200 rpm/min, 2500 rpm/min, 3000 rpm/min, 3500rpm/min, 4000 rpm/min, 4200 rpm/min, 4500 rpm/min, 5000 rpm/min, 5500rpm/min, 6000 rpm/min, 6500 rpm/min, 7000 rpm/min, 7500 rpm/min, 8000rpm/min, 8500 rpm/min, 9000 rpm/min, 9500 rpm/min, 10000 rpm/min, 11000rpm/min, 12000 rpm/min, 13000 rpm/min, 14000 rpm/min, 15000 rpm/min,16000 rpm/min, 17000 rpm/min, 18000 rpm/min, 19000 rpm/min or 20000rpm/min. Higher speed can be used if necessary.

Preferably, the nanomaterial is any one of or a mixture of at least twoof poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLA),polycaprolactone (PCL), polybutylene succinate, polyaniline,polycarbonate, poly(glycolide-co-lactide) orpoly(glycolide-co-caprolactone).

Preferably, the solvent is any one of or a mixture of at least two ofacetone, butanone, methanol, ethanol or isopropanol.

Preferably, the collection of the nanomaterial comprises: collecting theprepared nanomaterial by centrifugation, and then washing thenanomaterial by resuspending in deionized water twice. Thecentrifugation can be carried out at a rotation speed of 8000-15000rpm/min, for example, the rotation speed can be 8000 rpm/min, 9000rpm/min, 10000 rpm/min, 11000 rpm/min, 12000 rpm/min, 13000 rpm/min,14000 rpm/min, 14500 rpm/min., 14800 rpm/min, 15000 rpm/min. Higherspeeds can be used if necessary. Nanomaterials (nanoparticles) can becollected or further purified by other methods. The nanoparticles mayhave an average particle size as described above.

Preferably, the activation of the nanomaterial comprises: activating thenanomaterial for 0.5 to 5 hours by using a mixed solvent of 1 to 10mg/mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDS)and N-hydroxysuccinimide (NHS) at room temperature.

In the step (2) of the invention, the connection comprises: collectingthe activated nanomaterial by centrifugation, and then washing thenanomaterial once with the connecting reaction solution, adding amixture of the first antibody moiety and the second antibody moiety tobe connected in an equal volume into the connecting reaction solution,and then resuspending the nanomaterial with the connecting reactionsolution containing the first antibody moiety and the second antibodymoiety and conducting the connecting reaction for 0.5 to 5 hours at roomtemperature. After the reaction, the nanomaterial is collected bycentrifugation. The nanomaterial is washed twice in Dulbecco's phosphatebuffer saline (D-PBS), and then resuspended in D-PBS and stored at 4° C.Other methods of activating the nanomaterial can be employed.

The method for preparing the multi-specific binding conjugate of thepresent invention specifically includes the following steps:

(1) preparing a nanomaterial: completely dissolving the nanomaterial inacetone at a concentration of 5 to 30 mg/mL, and adding the solution ofthe nanomaterial in acetone to the deionized water in 1:4 v/v of acetoneand deionized water with magnetic stirring at 500 to 1500 rpm/min, toform a uniform emulsion, and then continuing to stir until thevolatilization of acetone;

(2) collecting the nanomaterial: collecting the prepared nanomaterial bycentrifugation at 8000 to 15000 rpm/min, and then resuspending indeionized water, which processes are repeated twice for washing thenanomaterial;

(3) activating the nanomaterial: activating the nanomaterial by using amixed solvent of 1 to 10 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide at room temperature for 0.5 to 5 hours;

(4) connecting the nanomaterial with antibodies: collecting theactivated nanomaterial by centrifugation, and then washing thenanomaterial once with 0.1 M D-PBS at pH=8.0, and adding a mixture ofthe first antibody moiety and the second antibody moiety to be connectedin an equal volume into the connecting reaction solution, and thenresuspending the nanomaterial with the connecting reaction solutioncontaining the first antibody moiety and the second antibody moiety andconducting the connecting reaction for 0.5 to 5 hours at roomtemperature. After the reaction, the nanomaterial is collected bycentrifugation. The nanomaterial is washed twice in D-PBS, and thenresuspended in D-PBS and stored at 4° C.

Pharmaceutical Composition

The invention also relates to a pharmaceutical composition comprisingthe multispecific binding conjugate. The pharmaceutical composition mayalso comprise a leukocyte, such as T cell, NK cell.

The pharmaceutical composition herein may be formulated using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate the entry of the active agent into thepharmaceutically acceptable formulation. Proper formulation depends onthe route of administration chosen. For example, an overview ofpharmaceutical composition can be found, for example, in Ansel et al,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th edition (Lea& Febiger 1990); and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th edition (Mack Publishing Company 1990) and its revised edition. Thepharmaceutical composition disclosed herein may further comprise one ormore diluents, excipients or carriers that are pharmaceuticallyacceptable. The pharmaceutical composition may comprise a drug orpharmaceutical agent, a carrier, an adjuvant such as a preservative, astabilizer, a wetting or emulsifying agent, a dissolution promoter, asalt for regulating the osmotic pressure, and/or a buffer.

In addition, the pharmaceutical composition may also contain othertherapeutically valuable substances, such as one or more othertherapeutic agents for treating the disease. For example, othertherapeutic agents may be other agents for treating cancer, such ascyclophosphamide, etoposide, carmustine, vincristine, etc., and may alsobe antibodies used to treat cancers, antibody-drug conjugates (ADC),interferons and/or checkpoint inhibitor antibodies.

Therapeutic Treatment

Various embodiments are directed to methods of treating a disease orcondition (e.g., cancer) in a subject, such as a mammal, including ahuman, domesticated or companion animals, such as a dog and a cat,comprising administering to the subject a therapeutically effectiveamount of the conjugate or pharmaceutical composition described herein.

The conjugate or pharmaceutical composition described herein may beformulated to be administered subcutaneously or even by other parenteralroutes, such as intravenously, intramuscularly, intraperitoneally orintravascularly, to a mammal via, for example, bolus injection orcontinuous infusion for intravenous administration. Preferably, theconjugate or pharmaceutical composition is infused for a period of lessthan about 4 hours, and more preferably for a period of less than about3 hours. For example, the first bolus can be infused over 30 minutes,preferably even 15 minutes, and the remainder is infused over the next 2to 3 hours. The injectable preparation may be provided in unit dosageform, for example, in ampoules or in multi-dose containers, with apreservative added. The composition may take such forms as a suspension,solution or emulsion in oily or aqueous vehicle, and may contain aformulating agent such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in the form of apowder which is reconstituted with a suitable vehicle, such as sterilepyrogen-free water, before use.

Additional pharmaceutical methods can be employed to control theduration of action of the therapeutic conjugate, pharmaceuticalcomposition, and/or other therapeutic agents. Controlled releaseformulation can be prepared by using a polymer to complex or adsorb theagent to be administered. For example, a biocompatible polymer includespoly(ethylene-vinyl acetate) copolymer matrix and polyanhydridecopolymer matrix of stearic acid dimers and sebacic acid. Sherwood etal, Bio/Technology 10: 1446 (1992). The rate of release from such amatrix depends on the molecular weight of the therapeutic agent, theamount of agent within the matrix, and the size of the dispersedparticles. Saltzman et al, Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th edition (Lea& Febiger 1990); and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th edition (Mack Publishing Company 1990) and its revised version.

More generally, the dosage of the conjugate administered to a human willvary depending on factors such as the age, weight, height, sex, generalmedical condition, and past medical history of the patient. It may bedesirable to provide the recipient with a conjugate dose as a singleintravenous infusion in the range of about 0.1 μg/kg to 25 mg/kg, bytotal amount of the first binding moiety and the second binding moietyin the multispecific binding conjugate, but lower or higher doses canalso be administered, as the case may be. For example, for a 70 kgpatient, a dose of 0.1 μg/kg-20 mg/kg is 0.7 μg-1400 mg. The dose can berepeated as needed, for example, once a week for 4 to 10 weeks, once aweek for 8 weeks or once a week for 4 weeks. In maintenance therapy, itcan also be administered at a lower frequency, if desired, such as everyother week for several months, or monthly or quarterly for many months.It is also possible to administer 2, 3, 4, 5 or 6 times continuously foreach course of treatment, for example, at an interval of about 10 days,15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50, 55days or 60 days or more after continuous administration for the nextcourse of treatment.

The conjugate can be administered with an effector cell such asleukocyte (such as T cell, NK cell), for example, via intravenousreinfusion. When the conjugate is administered with the effector cell,for example, it is performed once a week for 4-10 weeks, once weekly for8 weeks or once weekly for 4 weeks. In maintenance therapy, theconjugate and effector cell can also be administered at a lowerfrequency if desired, such as every other week or weeks, for severalmonths, or monthly or quarterly for many months. It is also possible toadminister 2, 3, 4, 5 or 6 times continuously for each course oftreatment, for example, at an interval of about 10 days, 15 days, 20days, 25 days, 30 days, 35 days, 40 days, 45 days, 50, 55 days or 60days or more after continuous administration for the next course oftreatment.

In general, the dosage of other therapeutic agents for humanadministration will vary depending on factors such as the age, weight,height, sex, general medical condition, and past medical history of thepatient. It may be desirable to provide the recipient with a conjugatedose as a single intravenous infusion in the range of about 1 mg/kg to25 mg/kg, but lower or higher doses may also be administered, asappropriate. For example, for a 70 kg patient, the dose of 1-20 mg/kg is70-1,400 mg. The dose can be repeated as needed, for example, once aweek for 4-10 weeks, once a week for 8 weeks or once a week for 4 weeks.In maintenance therapy, it can also be administered at a lowerfrequency, if desired, such as every other week for several months, ormonthly or quarterly for many months.

Other therapeutic agents can be administered in one dose every 2 weeksor 3 weeks, for a total of at least 3 doses repeated. Alternatively, thecombination can be administered twice a week for 4 to 6 weeks. If thedose is reduced to approximately 200-300 mg/m² (340 mg per dose for 1.7m patients or 4.9 mg/kg for 70 kg patients), it may be administered onceor even twice a week for 4 to 10 weeks. Alternatively, the time courseof administration can be reduced, i.e., once every 2 or 3 weeks for 2-3months. However, it has been determined that even higher doses can beadministered by slow i.v. infusion, such as 20 mg/kg once a week or onceevery 2-3 weeks, for repeated dosing cycles. The time course ofadministration can optionally be repeated at other intervals, and thedosage can be administered by various parenteral routes with appropriateadjustments in dosage and time course.

In a preferred embodiment, the conjugate or pharmaceutical compositiondescribed herein is useful in the treatment of cancer therapy. Examplesof cancer include, but are not limited to, carcinoma, lymphoma,glioblastoma, melanoma, sarcoma and leukemia, myeloma or lymphoidmalignancy. More specific examples of the cancer are described below andinclude: squamous cell carcinoma (e.g., epithelial squamous cellcarcinoma), Ewing's sarcoma, Wilms' tumor, astrocytoma, lung cancer(including small cell lung cancer, non-small cell lung cancer, lungadenocarcinoma and lung squamous cell carcinoma), peritoneal cancer,hepatocellular cancer, gastric cancer or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma multiforme,cervical cancer, ovary cancer, liver cancer, bladder cancer,hepatocellular cancer, hepatocellular carcinoma, neuroendocrine tumor,medullary thyroid carcinoma, thyroid differentiation cancer, breastcancer, ovarian cancer, colon cancer, rectal cancer, endometrial canceror uterine cancer, salivary gland cancer, renal or kidney cancer,prostate cancer, vulva cancer, anal cancer, penile cancer, and head andneck cancer. The term “cancer” includes primary malignant cell or tumor(e.g., a tumor where cells have not migrated to a site in a subjectother than the original malignant disease or tumor site) and secondarymalignant cells or tumors (e.g., a tumor produced by metastasis, i.e.,malignant cells or tumor cells migrating to a secondary site differentfrom the original tumor site). Cancers that benefit from the therapeuticmethod of the invention are directed to cells that express, overexpress,or aberrantly express IGF-IR.

Other examples of the cancer or malignant disease include, but are notlimited to, acute childhood lymphoblastic leukemia, acute lymphoblasticleukemia, acute lymphocytic leukemia, acute myeloid leukemia,adrenocortical carcinoma, adult (primary) hepatocellular carcinoma,adult (primary) liver cancer, adult acute lymphocytic leukemia, adultacute myeloid leukemia, adult Hodgkin's lymphoma, adult lymphocyticlymphoma, adult non-Hodgkin's lymphoma, adult primary liver cancer,adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignantdisease, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer,bone cancer, brain stem glioma, brain tumor, breast cancer, renal pelvisand ureteral cancer, central nervous system (primary) lymphoma, centralnervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma,cervical cancer, children (primary) hepatocellular carcinoma, children(primary) liver cancer, children acute lymphoblastic leukemia, childrenacute myeloid leukemia, children brain stem glioma, children cerebellarastrocytoma, children cerebral astrocytoma, children extracranialblastoma, Hodgkin's disease in children, Hodgkin's lymphoma in children,hypothalamic and visual pathway glioma in children, lymphoblasticleukemia in children, medulloblastoma in children, non-Hodgkin'slymphoma in children, pineal gland and supratentorial primitiveneuroectodermal tumor in children, children primary liver cancer,childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhoodvisual pathway and hypothalamic glioma, chronic lymphocytic leukemia,chronic myeloid leukemia, colon cancer, cutaneous T-cell lymphoma,endocrine islet cell carcinoma, endometrial cancer, ependymoma,epithelial cancer, esophageal cancer, Ewing's sarcoma and relatedtumors, exocrine pancreatic cancer, extracranial blastoma, extragonadalblastoma, extrahepatic cholangiocarcinoma, eye cancer, female breastcancer, Gaucher's disease, gallbladder cancer, stomach cancer, benigngastrointestinal tumor, gastrointestinal tumor, blastocytoma,gestational trophoblastic cell tumor, hairy cell leukemia, head and neckcancer, hepatocellular carcinoma, Hodgkin's lymphoma,hypergammaglobulinemia, hypopharyngeal carcinoma, intestinal cancer,intraocular melanoma, islet cell carcinoma, islet cell pancreaticcancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and mouthcancer, liver cancer, lung cancer, lymphoproliferative disorders,macroglobulinemia, male breast cancer, malignant mesothelioma, malignantthymoma, medulloblastoma, melanoma, mesothelioma, metastatic primaryoccult squamous neck cancer, metastatic primary squamous neck cancer,metastatic squamous neck cancer, multiple myeloma, multiplemyeloma/plasma neoplasms, myelodysplastic syndrome, myeloid leukemia,myelogenous leukemia, myeloproliferative disorders, nasal and paranasalsinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin'slymphoma, non-melanoma skin cancer, non-small cell lung cancer,metastatic primary occult squamous neck cancer, oropharyngeal cancer,osteosarcoma/malignant fibrosarcoma, osteosarcoma/malignant fibroushistiocytoma, osteosarcoma/malignant fibrous histiocytoma in bone,ovarian epithelial carcinoma, ovarian blastoma, ovarian low malignantpotential tumor, pancreatic cancer, diseased proteinemia, polycythemiavera, parathyroid carcinoma, penile cancer, pheochromocytoma, pituitarytumor, primary central nervous system lymphoma, primary liver cancer,prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis andureteral cancer, retinoblastoma, rhabdomyosarcoma, salivary glandcancer, sarcoidosis sarcoma, Sezary syndrome, skin cancer, small celllung cancer, small bowel cancer, soft tissue sarcoma, squamous neckcancer, gastric cancer, supratentorial primitive neuroectodermal andpineal tumor, T cell lymphoma, testicular cancer, thymoma, thyroidcancer, renal pelvis and ureteral transitional cell carcinoma,transitional renal pelvis and ureteral cancer, trophoblastic tumor,ureter and renal pelvic cell carcinoma, ureteral cancer, uterine cancer,uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma,vulva cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease other than a neoplasma located in theorgan system listed above.

The conjugate, composition, and method described and claimed herein canbe used to treat malignant or pre-malignant condition and to preventprogression to a neoplastic or malignant state, including but notlimited to those described above. It is indicated that such use isrelated to conditions that are known or suspected to progress in advanceinto a neoplasma or cancer, especially in the case of non-neoplasticcell growth that has occurred from hyperplasia, metaplasia or, mostparticularly, dysplasia. For a review of such abnormal growthconditions, see Robbins and Angell, BASIC PATHOLOGY, 2nd ed., W.B.Saunders Co., Philadelphia, pp. 68-79 (1976).

Dysplasia is often a precursor to cancer and is primarily found in theepithelium. It is the most disordered form of non-neoplastic cell growthand involves the loss of individual cell consistency and cell structureorientation. In the presence of chronic irritation or inflammation,dysplasia characteristically occurs. Dysplastic disorders that can betreated include, but are not limited to, non-perspiring ectodermaldysplasia, anterior dysplasia, asphyxiating thoracic dysplasia,atrial-finger dysplasia, bronchopulmonary dysplasia, cerebral dysplasia,cervical dysplasia, cartilage ectodermal dysplasia, clavicular skulldysplasia, congenital ectodermal dysplasia, skull diaphysis dysplasia,cranium carpus tarus dysplasia, skull metaphyseal dysplasia, dentinedysplasia, diaphysis dysplasia, ectodermal dysplasia, dental enameldysplasia, cerebral eyeball dysplasia, lateral epiphyseal dysplasia,multiple epiphyseal dysplasia, punctate epiphyseal dysplasia, epithelialdysplasia, facial phalangeal genital dysplasia, familial jaw fibrousdysplasia, familial white wrinkle dysplasia, fibromuscular dysplasia,bone fibrous dysplasia, vigorous bone dysplasia, hereditary renalretinal dysplasia, perspiring ectodermal dysplasia, hypohidroticectodermal dysplasia, lymphopenia thymic dysplasia, mammary glanddysplasia, mandibular face dysplasia, metaphyseal dysplasia, Montini'sdysplasia, monofibrillar bone dysplasia, mucous epithelial dysplasia,multiple epiphyseal dysplasia, ocular aural vertebral dysplasia, oculardental phalangeal dysplasia, ocular vertebral dysplasia, odontogenicdysplasia, ocular mandibular dysplasia, root tip periodontal cementaldysplasia, multifibrous bone dysplasia, pseudoachondroplasia vertebralepiphyseal dysplasia, retinal dysplasia, septal-ocular dysplasia,vertebral epiphyseal dysplasia and Cerebroventricular radial dysplasia.

Other pre-neoplastic disorders that can be treated include, but are notlimited to, benign abnormal proliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas and esophageal dysplasia), leukoplakia, keratosis, Bowen'sDisease, farmer's skin, solar cheilitis and solar keratosis.

In a preferred embodiment, the method of the invention is used toinhibit cancers, particularly the growth, progression and/or metastasisof the cancers listed above.

Other hyperproliferative diseases, disorders, and/or conditions include,but are not limited to, progression and/or metastasis of malignantdiseases and related conditions, such as leukemia (including acuteleukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemia (e.g., chronic myeloid(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors, including but not limited to sarcoma and carcinoma,such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma,osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma,lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor,mesothelioma, Ewing's Tumor, leiomyosarcoma, rhabdomyosarcoma, coloncancer, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,sweat gland cancer, sebaceous gland cancer, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,bronchial carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma,choriocarcinoma, seminoma, embryonal cancer, Wilms' tumor, cervicalcancer, testicular cancer, lung cancer, small cell lung cancer, bladdercancer, epithelial cancer, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pineal gland tumor, hemangioblastoma,acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastomaand retinoblastoma.

Kit

Various embodiments may involve a kit containing a component suitablefor treating or diagnosing diseased tissue of a patient. An exemplarykit can contain one or more conjugates, leukocytes, and/or othertherapeutic agents as described herein. If a composition containingmultiple components for administration is not formulated for deliveryvia the digestive tract, such as by oral delivery, a device capable ofdelivering the components of the kit by some other routes may beincluded. One type of device for applications such as parenteraldelivery is a syringe for injecting a composition into a subject. Aninhalation device can also be used. In certain embodiments, thetherapeutic agent can be provided in the form of a prefilled syringe orautoinjector pen containing a sterile liquid formulation or alyophilized formulation.

The components of kit can be packaged together or separated into two ormore containers. In some embodiments, the container can be a vialcontaining a sterile lyophilized formulation of a composition suitablefor reconstitution. The kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, bags,trays, boxes, tubes, and the like. The components of kit can beaseptically packaged and maintained in a container. Another componentthat can be included is the insert or leaflet for the kit.

EXAMPLE

The following examples are provided to illustrate the invention but notto limit the claims of the invention.

Example 1. Preparing Bispecific Binding ConjugateAnti-CD56-PLGA-Anti-CD20

A bispecific binding conjugate anti-CD56-PLGA-anti-CD20 having ananti-CD56 antibody and an anti-CD20 antibody coupled to differentnanoparticles (PLGA, PLA and PCL) was prepared.

The specific method for preparing the bispecific binding conjugateanti-CD56-PLGA-anti-CD20 is as follows:

(1) preparing a PLGA nanoparticle: completely dissolving the PLGA inacetone at a concentration of 5 mg/mL, and adding the solution of PLGAin acetone into deionized water in a volume ratio of 1:4 of acetone anddeionized water with magnetic stirring at 1000 rpm/min, to form auniform emulsion, and then continuing to stir until volatilization ofacetone;

(2) collecting the PLGA nanoparticle: collecting the preparednanoparticle with larger particle size by centrifugation at 8000 rpm/minfor 10 min; then collecting the prepared nanoparticle with smallerparticle size by centrifugation at 15000 rpm/min for 10 min, discardingthe nanoparticle with larger particle size and resuspending thenanoparticle with smaller particle size in deionized water, andrepeating the process twice to wash the nanoparticle. The nanoparticlewith smaller particle size is used in the process as follows;

(3) activating the PLGA nanoparticle: using a mixed solvent of 5 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide at room temperature to activate the PLGAnanoparticle for 1 h;

(4) connecting the PLGA nanoparticle to antibodies: collecting theactivated nanomaterial by centrifugation, and then washing thenanomaterial once with 0.1 M D-PBS at pH=8.0, mixing the anti-CD56monoclonal antibody (mAb) and the anti-CD20 mAb (both monoclonalantibodies are purchased from Tong Li Hai Yuan Biological Company) to beligated in an equal amount and adding them to the ligation reactionsolution, then resuspending the nanomaterial with the connectingreaction solution containing the anti-CD56 mAb and the anti-CD20 mAb,and conducting the connecting reaction for 0.5 h at room temperature.After the reaction, the nanomaterial is collected by centrifugation. Thenanomaterial is washed twice in D-PBS, and then resuspended in D-PBS andstored at 4° C.

The specific method for preparing the bispecific binding conjugateanti-CD56-PLA-anti-CD20 is as follows:

(1) preparing a PLA nanoparticle: completely dissolving PLA in methanolat a concentration of 15 mg/mL, adding the solution of PLA in methanolinto deionized water in a volume ratio of 1:4 of methanol and deionizedwater with magnetic stirring at 500 rpm/min, to form a uniform emulsion,and then continuing to stir until volatilization of methanol;

(2) collecting the PLA nanoparticle: collecting the preparednanoparticle by centrifugation at 10000 rpm/min for 10 min, and thenresuspending the nanoparticle in deionized water, and repeating theprocess twice to wash the nanoparticle;

(3) activating the PLA nanoparticle: using a mixed solvent of 1 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide at room temperature to activate the PLAnanoparticles for 0.5 h

(4) connecting the PLA nanoparticle to antibodies: collecting theactivated nanomaterial by centrifugation, then washing the nanomaterialonce with 0.1 M D-PBS at pH=8.0, mixing the anti-CD56 mAb and theanti-CD20 mAb to be ligated in an equal amount and adding them to theligation reaction solution, resuspending the nanomaterial with theconnecting reaction solution containing the anti-CD56 mAb and theanti-CD20 mAb, and conducting the connecting reaction for 0.5 h at roomtemperature. After the reaction, the nanomaterial is collected bycentrifugation and washed twice in D-PBS, and then resuspended in D-PBSand stored at 4° C.

The specific method for preparing the bispecific binding conjugateanti-CD56-PCL-anti-CD20 is as follows:

(1) preparing a PCL nanoparticle: completely dissolving PCL in isopropylalcohol at a concentration of 30 mg/mL, adding the solution of PCL inisopropyl alcohol into deionized water in a volume ratio of 1:4 ofisopropyl alcohol and deionized water with magnetic stirring at 1500rpm/min, to form a uniform emulsion, and then continuing to stir untilvolatilization of isopropyl alcohol;

(2) collecting the PCL nanoparticle: collecting the preparednanoparticle by centrifugation at 15000 rpm/min, and then resuspendingthe nanoparticle in deionized water, which processes are repeated twiceto wash the nanoparticle;

(3) activating the PCL nanoparticle: using a mixed solvent of 10 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide at room temperature to activate the PCLnanoparticle for 2 h;

(4) connecting the PCL nanoparticle to antibodies: collecting theactivated nanomaterial by centrifugation, then washing the nanomaterialonce with 0.1 M D-PBS at pH=8.0, mixing the anti-CD56 mAb and theanti-CD20 mAb to be ligated in an equal amount and adding them to theligation reaction solution, resuspending the nanomaterial with theconnecting reaction solution containing the anti-CD56 mAb and theanti-CD20 mAb, and conducting the connecting reaction for 3 h at roomtemperature. After the reaction, the nanomaterial is collected bycentrifugation and washed twice in D-PBS, and then resuspended in D-PBSand stored at 4° C.

Example 2. Preparing Other Bispecific Binding Conjugates

Other bispecific binding conjugates i.e. anti-CD16-PLGA-anti-MUC1,anti-CD56-PLGA-anti-MUC1, anti-CD16-PLGA-anti-CD19,anti-CD56-PLGA-anti-CD19, anti-CD16-PLGA-anti-CD20 andanti-CD56-PLGA-anti-CD20 were prepared in a manner similar to thatdescribed in Example 1. The antibodies used therein are all commerciallyavailable monoclonal antibodies.

In addition, anti-CD56-PLA-anti-CD19, anti-CD56-PCL-anti-CD19,anti-CD16-PLA-anti-CD20, anti-CD16-PCL-anti-CD20,anti-CD16-PLA-anti-MUC1 and anti-CD16-PCL-anti-MUC1 were also preparedin a manner similar to that described in Example 1.

Example 3. Efficacy of Bispecific Binding Conjugates on Killing CancerCells

The ability of the bispecific binding conjugates prepared in Example 1and Example 2 to kill tumor cells was evaluated.

Specifically, 5000 target cells per well were cultured for 12 h in96-well plates and then the initial medium was discarded. Thecytokine-free X-vivo 15 medium (purchased from lonza) was used to adjustthe density of NK cells (induced by peripheral blood mononuclear cellsfrom healthy blood donors), so that number of NK cells in a volume of100 μl reaches 4 times larger than that of the target cells (effector totarget ratio is 4:1). 100 μl suspension of NK cells was added in acancer cell culture plate, and 10 μl prepared bispecific bindingconjugate (bispecific binding conjugate content: 0.2 mg, with a totalamount of monoclonal antibodies 0.2 μg) was added, incubated for 8 h inan incubator, and then CCK-8 reagent was added, incubated according tothe reagent instruction. The absorbance at 450 nm was measured using amicroplate reader. Statistical analysis of the data was performed,according to the following formula to calculate killing rate of DC-CIKcells on cancer cells.

Killing rate=[1−(experimental group−effector control group)/(targetcontrol group−blank control group)]×100%

The blank control group represents the added medium; the target controlgroup represents the added target cell+medium; the effector controlgroup represents the added effector cell+medium; the experimental grouprepresents the effector cell+target cell+medium+bispecific bindingconjugate.

The results are shown in Tables 1-2 and FIGS. 2-7.

TABLE 1 Ability of bispecific binding conjugates to kill tumor cells(effector-target ratio of 4:1) Killing rate Effector Experimental groupcontrol (NK + conjugate) group Anti-CD56-PLGA- Anti-CD16-PLGA- Cancercell (NK) anti-CD20 anti-MUC1 HKC 7.44% 9.23% 11.23% A549 9.58% 81.91%89.91% HepG2 14.68% 89.36% 80.36% HT29 15.67% 92.47% 82.47% MCF-7 19.57%77.23% 87.23% AGS 12.46% 93.21% 84.21% Hela 15.76% 79.64% 85.64% 563713.57% 80.74% 87.74% OS-RC-2 10.62% 94.47% 80.47% Note: HKC - humanembryo kidney epithelial cells; A549 - human non-small cell lung cancercells; HepG2 - human liver cancer cells; HT29 - human colon cancercells; AGS - human gastric adenocarcinoma cells; Hela - human cervicalcancer cells; 5637 - human bladder cancer cells; OS-RC-2 - human kidneycancer cells.

TABLE 2 Ability of bispecific binding conjugates to kill tumor cells(effector-target ratio of 4:1) Killing rate Experimental group (NK +conjugate) Effector Anti-CD16- Anti-CD56- Anti-CD16- Anti-CD56- Cancercontrol PLGA-anti- PLGA-anti- PLGA-anti- PLGA-anti- cell group (NK) CD19CD19 CD20 CD20 Raji 12.59% 79.91% 83.85% 91.54% 92.94% RAMOS 17.67%81.36% 90.27% 94.38% 85.37% Note: Raji - black Burkitt lymphoma cells;RAMOS - human B lymphocytoma cells. Anti-CD56-PLA-anti-CD19 andanti-CD56-PCL-anti-CD19 showed a killing rate greater than 83% for blackBurkitt lymphoma cells and human B lymphocytoma cells (specific data notshown).

Anti-CD16-PLA-anti-CD20 and anti-CD16-PCL-anti-CD20 showed a killingrate greater than 83% for black Burkitt lymphoma cells and human Blymphocytoma cells (specific data not shown).

Anti-CD16-PLA-anti-MUC1 and anti-CD16-PCL-anti-MUC1 showed a killingrate greater than 95% for human non-small cell lung cancer cells;greater than 92% for human bladder cancer cells and human breast cancercells; nearly 85% for human cervical cancer cells and human gastricadenocarcinoma cells; greater than 80% for human colon cancer cells,human kidney cancer cells and human liver cancer cells.

Example 4. Experiment of Inhibiting Tumor In Vitro

Anti-CD3 and/or anti-CD16 and anti-MUC1 bispecific or trispecificbinding conjugates connected by PLGA (i.e. anti-CD3-PLGA-anti-MUC1,anti-CD16-PLGA-anti-MUC1 or anti-CD3 plus anti-CD16-PLGA-anti-MUC1,hereinafter referred to as CD3-MUC1 group, CD16-MUC1 group andCD3/CD16-MUC1 group, respectively) were prepared according to themethods of the previous Examples, wherein the antibodies used were allcommercially available.

The ability of the bispecific or trispecific binding conjugates toassist NK to kill A549 cells was evaluated. Specifically, 50 ml ofperipheral blood of healthy human was collected, monocytes (PBMC) wereisolated, and then NK cytokines were added for induction and expansionculture. The percentage of NK cells was measured by culturing cellsuntil day 21. The results were as follows: the ratio of NK cells was86.9%, and the ratio of CD3+ T cells was 10.2% (see FIG. 8). 5000 A549cells as target cells were cultured in each well for 12 h in 96-wellplates and then the initial medium was discarded. The cytokine-freeX-vivo 15 medium was used to adjust the density of NK cells, so that thenumber of NK cells in a volume of 100 μl was 4 times larger than that ofthe target cells (effector-target ratio is 4:1). 100 μl suspension of NKcells was added in a cancer cell culture plate, and 10 μl preparedbispecific binding conjugates (the content of bispecific bindingconjugate was 0.2 mg, with a total amount of monoclonal antibodies 0.2μg) were added, incubated for 8 h in an incubator, and then CCK-8reagent added, incubated according to the reagent instructions. Theabsorbance at 450 nm was measured using a microplate reader. Statisticalanalysis of the data was performed, according to the following formulato calculate killing rate of NK cells on cancer cells.

Killing rate=[1−(experimental group−effector control group)/(targetcontrol group−blank control group)]×100%

The results of the experiment showed (see FIG. 9): NK, CD3 monoclonalantibody+NK, CD16 monoclonal antibody+NK, MUC1 monoclonal antibody+NKshowed a certain effect, and the statistical results showed thatcompared with the control group, the tumor inhibition rates were 26.1%,33.02%, 26.22%, and 37.51%, respectively, and there was no significantdifference between the groups. The tumor inhibition rates of CD3-MUC1,CD16-MUC1, CD3/CD16-MUC1 groups to assist NK cells were 71.02%, 81.84%,and 90.91%, respectively, which showed good efficacy, withCD3/CD16-MUC1>CD16-MUC1>CD3-MUC1 and also closely related to thecellular components in NK cells. NK generally expresses CD16, and theproportion of NK cells is much larger than that of CD3+ T cells.Therefore, it is appropriate to use CD16 as the recognition site of NK.Because the NK cell population currently induced in vitro has NK cellsat a ratio of about 86.9% and some other effector cells (including 10.2%of CD3+ T cells), the anti-tumor effect of CD16 and CD3 both of whichare connected is better.

Example 5. Experiment of Inhibiting Tumor In Vivo

The anti-tumor efficacy of the bispecific or trispecific bindingconjugates described in Example 4 was evaluated in vivo. Specifically,healthy Balb-c nude mice (clean grade, female, four weeks old, bodyweight 18-22 g, purchased from Beijing Vital Lihua Experimental AnimalTechnology Co., Ltd.) were inoculated with human lung adenocarcinomacell A549 were at oxter. After 3-4 weeks of inoculation, 90 nude micewith a tumor diameter of about 0.5×0.5 cm were randomly divided into 8groups for experiments (grouping is shown below).

Groups Experimental treatments Control normal saline 100 ul NK 1 × 10⁶NK cells CD3 1 × 10⁶ NK cells + CD3 mAb CD16 1 × 10⁶ NK cells + CD16 mAbMUC1 1 × 10⁶ NK cells + MUC1 mAb CD3-PLGA-MUC1 1 × 10⁶ NK cells +CD3-PLGA-MUC1 bispecific binding conjugate CD16-PLGA-MUC1 1 × 10⁶ NKcells + CD16-PLGA-MUC1 bispecific binding conjugate CD3/CD16-PLGA-MUC1 1× 10⁶ NK cells + CD3/CD16-PLGA- MUC1 trispecific binding conjugate

The mice were administered by tail vein injection on days 1, 2, and 3,respectively. Each experimental group was injected with 20 μl ofantibody or binding conjugate preparation each time, wherein thepreparation contained 12.5 μg of each monoclonal antibody, that is, 0.25μg of antibody per animal was injected in the monoclonal antibody group,0.50 μg of antibody per animal in total was injected in the bispecificbinding conjugate group, and 0.75 μg of antibody per animal in total wasinjected in the trispecific binding conjugate group. The control groupwas injected with the same volume of normal saline.

The volume of the recorded tumor was measured from the first day. Thetherapeutic effect of the bispecific/trispecific binding conjugates onthe tumor was calculated.

FIG. 10 shows the statistical changes in tumor volume.

The results of the first 22 days of the experiment showed that: NK, CD3monoclonal antibody+NK, CD16 monoclonal antibody+NK, MUC1 monoclonalantibody+NK showed a certain effect, and the current statistical resultsshowed that compared with the control group, the tumor inhibition ratewas 39.24%, 36.19%, 33.49%, and 37.01%, respectively, and there was nosignificant difference among the groups. The tumor inhibition rates ofCD3-MUC1, CD16-MUC1, CD3/CD16-MUC1 to assist NK cells were 81.22%,88.24%, and 95.15%, respectively, which showed good efficacy, withCD3/CD16-MUC1>CD16-MUC1>CD3-MUC1 and also closely related to thecellular components in NK cells. NK generally expresses CD16, and theproportion of NK cells is much larger than that of CD3+ T cells.Therefore, it is appropriate to use CD16 as the recognition site of NK.Because the NK cell population currently induced in vitro has NK cellsat a ratio of about 86.9% and some other effector cells (including 10.2%of CD3+ T cells), the anti-tumor effect of CD16 and CD3 both of whichare connected is better.

In accordance with the present disclosure, all of the conjugates,compositions, and methods disclosed and claimed herein can be made andused without undue experimentation. Although the compositions andmethods have been described in terms of the preferred embodiments, itwill be apparent to those skilled in the art that the conjugates,compositions and methods described herein as well as the steps or stepsequences in the methods can vary without departing from the concept,spirit and scope of the invention. More specifically, certain reagentsthat are chemically and physiologically related may be substituted forthe reagents described herein while achieving the same or similarresults. All such similar substitutions and modifications which areobvious to those skilled in the art, are within the spirit, scope andconcept of the invention as defined by the appended claims.

1. A multispecific binding conjugate comprising: a) at least one firstbinding moiety that binds to a receptor, co-receptor, antigen and/orcellular marker on a cytotoxic effector cell which is a leukocyte whichis selected from the group consisting of an NK cell and an NKT cell; andb) at least one second binding moiety that binds to a receptor,co-receptor, antigen and/or cellular marker on a target cell which is atumor cell; wherein the first binding moiety is an antibody or afragment thereof; wherein the first binding moiety and the secondbinding moiety are coupled to a nanoparticle which is preferably abiodegradable nanomaterial, more preferably any one of or a mixture ofat least two of polylactic acid-co-glycolic acid), polylactic acid,polycaprolactone, polybutylene succinate, polyaniline, polycarbonate,poly(glycolide-co-lactide) or poly(glycolide-co-caprolactone).
 2. Theconjugate according to claim 1 wherein the receptor, co-receptor,antigen and/or cellular marker on the NK cell is selected from the groupconsisting of CD16, CD56, CD57, ADAM17 and CD137; and b) the target cellis a diseased cell or pathogen.
 3. The conjugate according to claim 2,wherein the receptor, co-receptor, antigen and/or cell marker on thediseased cell is selected from the group consisting of: carbonicanhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3, A33antibody-specific antigen, AFP, ART-4, B7, Ba 733, BAGE, BrE3-antigen,CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4,CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46,CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a,CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154,CDC27, CDK-4/m, CDKN2A, CXCR4, CXCR7, CXCL12, HIF-1alpha, colon-specificantigen-p (CSAp), CEA(CEACAM5), CEACAM6, c-met, DAM, EGFR, EGFRvIII,EGP-1, EGP-2, ELF2-M, Ep-CAM, Fit-1, Flt-3, folate receptor, G250antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, human chorionic gonadotropin(HCG) and its subunit, HER2/neu, HMGB-1, hypoxia-inducible factor(HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R,IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17,IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen,KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitoryfactor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP,MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13,MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin,placental growth factor, p53, PLAGL2, prostate acid phosphatase, PSA,PRAME, PSMA, PIGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE,S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor,TNF-alpha, Tn antigen, Thomson-Friedrich antigen, tumor necrosisantigen, TROP-2, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen,complement factor C3, C3a, C3b, C5a, C5, angiogenesis marker, bc1-2,bc1-6, Kras, cMET, oncogene product.
 4. The conjugate according to claim2, wherein the receptor, co-receptor, antigen and/or cell marker on thediseased cell is a cell surface protein selected from the groupconsisting of cholecystokinin B Receptor, gonadotropin releasing hormonereceptor, somatostatin receptor 2, avb3 integrin, gastrin releasingpeptide receptor, neurokinin 1 receptor, melanocortin 1 receptor,neurotensin receptor, neuropeptide Y receptor and C-type lectin-likemolecule
 1. 5. The conjugate according to claim 1, wherein the firstbinding moiety binds to CD16 or CD56.
 6. (canceled)
 7. The conjugateaccording to claim 1, wherein the second binding moiety binds to anantigen selected from the group consisting of AFP, CD19, CD20, CD38,MUC1, EGFR and HER2/neu.
 8. The conjugate according to claim 1, whereinthe antibody is selected from the group consisting of hR1 (anti-IGF-1R),hPAM4 (anti-mucin), KC4 (anti-mucin), hA20 (anti-CD20), hA19(anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22),RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14(anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-TROP-2), hMN-3(anti-CEACAM6), CC49 (anti-TAG-72), J591 (anti-PSMA), D2/B (anti-PSMA),G250 (anti-carbonic anhydrase IX), infliximab (anti-TNF-α), certolizumabpegol (anti-TNF-α), adalimumab (anti-TNF-α), alemtuzumab (anti-CD52),bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33),ibritumomab (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20),tositumomab (anti-CD20), GA101 (anti-CD20), trastuzumab (anti-HER2/neu),tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab(anti-CD25), efalizumab (anti-CD1Ia), muromonab-CD3 (anti-CD3 receptor),natalizumab (anti-α4 integrin), BWA-3 (anti-histone H2A/H4), LG2-1(anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B),LG11-2 (anti-histone H2B), LG2-2 (anti-histone H2B), P4/D10 (anti-gp120)and omalizumab (anti-IgE).
 9. The conjugate according to claim 1,wherein the second antibody is selected from the group consisting ofhA19, hR1, hPAM4, hA20 (veltuzumab), hIMMU31, hLL1 (milatuzumab), hLL2(epratuzumab), hMu-9, hL243, hMN-14, hMN-15, hRS7 and hMN-3.
 10. Theconjugate according to claim 1, wherein the nanoparticle is poly(lacticacid-co-glycolic acid), polylactic acid and/or polycaprolactone.
 11. Theconjugate according to claim 1, wherein the conjugate further comprisesthe first binding moiety that binds to the receptor, co-receptor,antigen and/or cellular marker on the T cell.
 12. The conjugateaccording to claim 11, wherein the receptor, co-receptor, antigen and/orcellular marker on the T cell is selected from the group consisting ofCD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45,CD69 and CD90.
 13. A pharmaceutical composition comprising the conjugateof claim
 1. 14. The pharmaceutical composition according to claim 13further comprising leukocyte, the leukocyte comprising NK cell.
 15. Thepharmaceutical composition according to claim 13, further comprising anadditional therapeutic agent, preferably selected from the groupconsisting of an antibody, an antibody fragment, a drug, a toxin, anenzyme, a cytotoxic agent, an anti-angiogenic agent, pro-apoptoticagent, antibiotic, hormone, immunomodulator, cytokine, chemokine,antisense oligonucleotide, small interfering RNA (siRNA), boroncompound, and radioisotope.
 16. A method of treating a disease orcondition in a subject in need thereof, comprising administering to thesubject the multispecific binding conjugate of claim
 1. 17. The methodaccording to claim 16, wherein the disease or condition is cancer,preferably a cancer selected from the group consisting of non-Hodgkin'slymphoma, B cell lymphoma, B cell leukemia, T cell lymphoma, T cellleukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia,Burkitt's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acutemyelogenous leukemia, chronic myelogenous leukemia, multiple myeloma,glioma, Waldenstrom's macroglobulinemia, carcinoma, melanoma, sarcoma,glioma, skin cancer, oral cancer, gastrointestinal cancer, colon cancer,stomach cancer, lung cancer, breast cancer, ovarian cancer, prostatecancer, uterine cancer, endometrial cancer, cervical cancer, bladdercancer, pancreatic cancer, bone cancer, liver cancer, gallbladdercancer, kidney cancer, testicular cancer, epithelial cancer, colorectalcancer, stomach cancer, brain cancer, glioblastoma, pancreatic cancer,myeloid leukemia, cervical cancer, medullary thyroid carcinoma,astrocytoma, prostate cancer, bladder cancer, neuroendocrine cancer,gastrointestinal pancreatic tumor, exocrine pancreatic cancer and Ewingsarcoma.
 18. The method according to claim 16, further comprisingadministering to the subject an additional therapeutic agent, preferablyselected from the group consisting of an antibody, an antibody fragment,a drug, a toxin, an enzyme, a cytotoxic agent, an anti-angiogenic agent,pro-apoptotic agent, antibiotic, hormone, immunomodulator, cytokine,chemokine, antisense oligonucleotide, small interfering RNA (siRNA),boron compound, and radioisotope.